I am investigating how known changes in the primary structure of a protein affect the kinetic and equilibrium properties of protein folding reactions. Iso-1 and iso-2 cytochrome c's are the major focus of the work since these two proteins are particularly well suited for primary structure manipulations by genetics and recombinant DNA methods. Fast kinetics, stopped flow mixing and temperature jumps, will be used to determine which of the kinetic processes in folding of the wild-type protein are perturbed by changes in the primary structure of the mutant protein. Differences in stability of wild-type and mutant proteins will be measured by thermal and guanidine hydrochloride denaturation. Effects of mutations located within or at the interface between structural elements found in the native protein will be investigated. The focus will be on proteins with mutations at or near conserved sites. Interpretation of mutation-induced changes in the properties of folding depends critically on whether folding proceeds along similar pathways to similar final structures in mutant and wild-type proteins. Optical and functional assays for the presence of native or native-like species are proposed as a test of whether the final steps in the folding pathway of the wild-type protein are retained in refolding of mutant proteins. The structure and internal dynamics of folded mutant and wild-type proteins will be compared by proton NMR spectroscopy. Using J-echo difference spectroscopy to resolve and assign resonance from protons attached to 13C labeled aromatic side chains, ring flip rates will be measured and used as probes of the internal dynamics below and within the unfolding transition zone. Pseudo-contact paramagnetic shifts of assigned resonances will provide information on whether the mutant proteins have folded to native-like structures.